We have prepared N-tritioacetoxyphthalimide as a new tritioacetylating reagent for the labelling of peptides& amino acids and many other biologically important compounds. The only commercially available tritioacetylating reagent, tritiated acetic anhydride is volatile, moisture sensitive, and half of the radioactivity is unused in the acetylation reaction. We required a reagent free of these problems, and our exploratory studies led to the preparation of several precursors, as reported in previous years. In our final approach the synthesis of N-iodoacetoxyphthalimide was pursued. This reagent was synthesized using N-hydroxyphthalimide and iodoacetic acid in the presence of DCC. The crystalline product was isolated and the preliminary catalytic dehalogenation reaction with hydrogen gas and Pd/C was carried out, with routine monitoring by TLC. This reagent gave about 30% chemical yield of N-deuterioacetoxyphthalimide, and the deuterium content of the product was low (ca. 40% of theoretical). In order to improve the labelling reaction, we replaced the traditional catalytic dehalogenation approach by using radical dehalogenation techniques, relying on our newly developed tributyltin tritide reagent. In preliminary studies, the N-iodoacetoxyphthalimide was reacted with commercial tributyltin deuteride, and triethyl borane was used as radical initiator, in dry THF. The reaction generated the desired N-deuterioacetoxyphthalimide at 40% chemical yield, with 60% of the theoretical deuterium content. As an example of the selectivity of the new reagent, muramic acid, a component of N-acetyl muramyl dipeptide present in the bacterial cell wall, was N-deuteroacetylated without affecting the several hydroxyl functional groups present in the molecule. We should note that N-acetylation of compounds such as muramic acid with acetic anhydride gives products from intramolecular cyclization (lactams), not the desired acetylated products. NMR analysis of the purified product indicated that the amino acid was successfully N-deuterioacetylated by our new reagent. Encouraged by the selectivity of the reagent and the successful dehalogenation of the iodo precursor, tributyltin deuteride was prepared at the NTLF and quantitated by a GC method to give 67% yield and used in the radical dehalogenation reaction. The products were analyzed by HPLC, proton and deuterium NMR and revealed that the freshly generated TBTD works comparably to the commercial tributyltin deuteride. The next step was to tritiate the reagent by generating high specific activity tributyltin tritide under the same conditions as the deuteride. Several batches of the acetylation reagent were prepared and very carefully analyzed by radio-HPLC and tritium NMR each time. The proton NMR analysis showed the desired product was present with some byproducts that were formed during the radical dehalogenation reaction. The tin byproducts and other impurities were removed by dissolving the crude reaction mixture in acetonitrile and extraction of the impurities with hexane several times. The repeated synthesis of this reagent, followed by radio-HPLC and tritium NMR analyses gave an average chemical yield of 38-40% and specific activity in the range 13-18 Ci/mmole. To further demonstrate the applicability and selectivity of the tritioacetylating reagent two peptides were selected for N-acetylation ACTH (1-4) (H2N-Ser-Tyr-Ser-Met-OH) and Neurotensin (8-13) (H2N-Arg-Arg-Pro-Tyr-Ile-Leu-OH). None of the functional groups in these two peptides were protected before the acetylation. A sample of each peptide was acetylated with the non-radioactive reagent first and the products were analyzed by NMR and mass spectrometry to show very satisfactory results. ACTH was N-tritioacetylated at the N-terminal serine to give a specific activity of 18.3 Ci/mmole and the Neurotensin at the N-terminal arginine to furnish a chemical yield of 37% and specific activity of 11.3 Ci/mmole. Tritium NMR analyses confirmed the synthesis of the N-[3H]-acetylated ACTH and N-[3H]-acetylated Neurotensin. This project is now completed and a manuscript has been prepared for publication. As a part of this project we also investigated the deuterium labelling of bromoacetic acid by a catalytic dehalogenation method reported in the literature to find the extent of the deuterium labelling in the product, a-deuteriated sodium acetate. We have found 40% deuterium incorporation under the reported reaction conditions, compared to a published incorporation of 100% tritium. Further experiments are required to confirm our results,&to understand the error in the published work. As a prototypical acetylation reaction, we reacted the labelled sodium acetate with benzylamine at pH 5 in water with EDC as the coupling reagent, thus generating N-acetylbenzylamine. Further optimization of this reaction is also necessary.